Download Adoptive cell transfer as personalized immunotherapy for human

REVIEWS
Adoptive cell transfer as personalized
immunotherapy for human cancer
Steven A. Rosenberg* and Nicholas P. Restifo*
Adoptive cell therapy (ACT) is a highly personalized cancer therapy that involves
administration to the cancer-bearing host of immune cells with direct anticancer activity.
ACT using naturally occurring tumor-reactive lymphocytes has mediated durable, complete
regressions in patients with melanoma, probably by targeting somatic mutations
exclusive to each cancer. These results have expanded the reach of ACT to the treatment
of common epithelial cancers. In addition, the ability to genetically engineer lymphocytes
to express conventional T cell receptors or chimeric antigen receptors has further
extended the successful application of ACT for cancer treatment.
A
doptive cell therapy (ACT) has multiple
advantages compared with other forms of
cancer immunotherapy that rely on the
active in vivo development of sufficient
numbers of antitumor T cells with the functions necessary to mediate cancer regression. For
use in ACT, large numbers of antitumor lymphocytes (up to 1011) can be readily grown in vitro
and selected for high-avidity recognition of the
tumor, as well as for the effector functions required
to mediate cancer regression. In vitro activation
allows such cells to be released from the inhibitory factors that exist in vivo. Perhaps most importantly, ACT enables the manipulation of the host
before cell transfer to provide a favorable microenvironment that better supports antitumor immunity. ACT is a “living” treatment because the
administered cells can proliferate in vivo and
maintain their antitumor effector functions.
A major factor limiting the successful use of
ACT in humans is the identification of cells that
can target antigens selectively expressed on the
cancer and not on essential normal tissues. ACT
has used either natural host cells that exhibit
antitumor reactivity or host cells that have been
genetically engineered with antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs).
With the use of these approaches, ACT has mediated dramatic regressions in a variety of cancer
histologies, including melanoma, cervical cancer,
lymphoma, leukemia, bile duct cancer, and neuroblastoma. This Review will discuss the current state
of ACT for the treatment of human cancer, as
well as the principles of effective treatment that
point toward improvements in this approach.
A brief history of ACT
Very little was known about the function of T
lymphocytes until the 1960s, when it was shown
that lymphocytes were the mediators of allograft rejection in experimental animals. Attempts
to use T cells to treat transplanted murine tumors were limited by the inability to expand and
Surgery Branch, National Cancer Institute, Center for Cancer
Research, National Institutes of Health, 9000 Rockville Pike,
CRC Building, Room 3W-3940, Bethesda, MD 20892, USA.
*Corresponding author. E-mail: [email protected] (S.A.R.); restifo@
nih.gov (N.P.R.)
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manipulate T cells in culture. Thus, ACT used
transfer of syngeneic lymphocytes from rodents
heavily immunized against the tumor, and modest
growth inhibition of small established tumors
was observed (1, 2). In early preclinical studies,
the importance of host inhibitory factors was suggested by findings that lymphodepletion using
either chemotherapy or radiation before cell transfer enhanced the ability of transferred lymphocytes
to treat established tumors (3, 4).
The ability to use ACT was facilitated by the
description of T cell growth factor [interleukin-2
(IL-2)] in 1976, which provided a means to grow
T lymphocytes ex vivo, often without loss of effector functions (5). The direct administration of
high doses of IL-2 could inhibit tumor growth in
Reinfuse postlymphodepletion
Select and
expand to
1010 cells
mice (6), and studies in 1982 demonstrated that
the intravenous injection of immune lymphocytes
expanded in IL-2 could effectively treat bulky
subcutaneous FBL3 lymphomas (7). In addition,
administration of IL-2 after cell transfer could
enhance the therapeutic potential of these adoptively transferred lymphocytes (8). The demonstration in 1985 that IL-2 administration could result
in complete durable tumor regressions in some
patients with metastatic melanoma (9) provided
a stimulus to identify the specific T cells and their
cognate antigens involved in this cancer immunotherapy. Lymphocytes infiltrating into the stroma
of growing, transplantable tumors were shown to
represent a concentrated source of lymphocytes capable of recognizing tumor in vitro, and studies in
murine tumor models demonstrated that the adoptive transfer of these syngeneic tumor-infiltrating
lymphocytes (TILs) expanded in IL-2 could mediate
regression of established lung and liver tumors
(10). In vitro studies in 1986 showed that human
TILs obtained from resected melanomas contained
cells capable of specific recognition of autologous
tumors (11), and these studies led in 1988 to the
first demonstration that ACT using autologous
TILs could mediate objective regression of cancer in patients with metastatic melanoma (12).
Populations of TILs that grow from tumors
are generally mixtures of CD8+ and CD4+ T cells
with few if any major contaminating cells in
mature cultures. The ability of pure populations
of T lymphocytes to mediate cancer regression
in patients provided the first direct evidence that
T cells played a vital role in human cancer immunotherapy. However, responses were often of short
Excise
tumor
Plate
fragments
-
-
+
-
+
-
Assay for
specific tumor
recognition
Culture with
6000 IU/mL IL-2
Fig. 1. General schema for using the adoptive cell transfer of naturally occurring autologous TILs. The
resected melanoma specimen is digested into a single-cell suspension or divided into multiple tumor fragments
that are individually grown in IL-2. Lymphocytes overgrow, destroy tumors within 2 to 3 weeks, and generate
pure cultures of lymphocytes that can be tested for reactivity in coculture assays. Individual cultures are then
rapidly expanded in the presence of excess irradiated feeder lymphocytes, OKT3, and IL-2. By approximately
5 to 6 weeks after resecting the tumor, up to 1011 lymphocytes can be obtained for infusion into patients.
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C A NC E R IM M UN OL OG Y A N D I MM U NO TH E R AP Y
duration, and the transferred cells could rarely
be found in the circulation just days after administration. A critical improvement in the application of ACT to the treatment of human cancer was
reported in 2002, when it was shown that lymphodepletion using a nonmyeloablative chemotherapy
regimen administered immediately before TIL
transfer could lead to increased cancer regression,
as well as the persistent oligoclonal repopulation
of the host with the transferred antitumor lymphocytes (13). In some patients, the administered antitumor cells represented up to 80% of the CD8+ T
cells in the circulation months after the infusion.
Lymphocyte cultures can be grown from many
tumor histologies; however, melanoma appeared
to be the only cancer that reproducibly gave rise
to TIL cultures capable of specific antitumor recognition. The stimulus to more widely apply ACT
to treat multiple human cancers led to studies
of the genetic engineering of lymphocytes to
express antitumor receptors. Following mouse
models (14), it was shown for the first time in
humans in 2006 that administration of normal
circulating lymphocytes transduced with a retrovirus encoding a TCR that recognized the MART-1
melanoma-melanocyte antigen could mediate
tumor regression (15). Administration of lymphocytes genetically engineered to express a chimeric
antigen receptor (CAR) against the B cell antigen CD19 was shown in 2010 to mediate regression of an advanced B cell lymphoma (16). These
findings of the use of either naturally occurring
or genetically engineered antitumor T cells set
the stage for the extended development of ACT
for the treatment of human cancer.
tered for 5 days followed by cells and IL-2 given at
720,000 IU/kg to tolerance (Fig. 2). In a pilot study
in the Surgery Branch, National Cancer Institute
(NCI), objective cancer regressions by RECIST criteria (Response Evaluation Criteria in Solid Tumors)
were seen in 21 of 43 patients (49%), including 5
patients (12%) who underwent complete cancer
regression (13). When 200 or 1200 centigray (cGy;
1 Gy = 100 rads) total-body irradiation (TBI) was
added to the preparative regimen in pilot trials
of 25 patients each, objective response (OR) rates
34 complete responders thus far seen in the two
trials at the NCI, only one has recurred, and only
one patient with complete regression received more
than one treatment. The brain is not a sanctuary
site, and regression of brain metastases has been
observed (21). Prior treatment with targeted therapy using the Braf inhibitor vemurafenib (Zelboraf)
does not appear to affect the likelihood of having
an OR to ACT treatment in patients with melanoma. ACT can also be effective after other immunotherapies have failed. Of the 194 patients treated
Lymphodepletion prior to T cell transfer is followed
by immune reconstitution
Peripheral blood cell count
6000 cells per mm3
White blood
cell count
5000
Cyclophosphamide
4000
Absolute
neutrophil
count
Fludarabine
3000
T cell
transfer
2000
IL-2
1000
0
-10
-5
0
Absolute
lymphocyte
count
5
10
15
20
25
Days from cell infusion
ACT using TILs is an effective
immunotherapy for patients
with metastatic melanoma
Fig. 2. A substantial increase in cell persistence and the incidence and duration of clinical responses is observed when patients received a lymphodepleting preparative regimen before the
cell infusion. The most frequently used lymphodepleting preparative regimen consists of 60 mg/kg
cyclophosphamide given for 2 days and 25 mg/m2 fludarabine administered over 5 days, followed by
T cells and IL-2 administration.
Adoptive cell therapy using autologous TILs is
the most effective approach to induce complete
durable regressions in patients with metastatic
melanoma (Table 1). The general approach for
growing and administering human TILs is shown
in Fig. 1. The resected melanoma specimen is
digested into a single-cell suspension or divided
into multiple tumor fragments that are individually grown in IL-2. Lymphocytes overgrow, destroy tumors within 2 to 3 weeks, and give rise to
pure cultures of lymphocytes that can be tested
for reactivity against tumors, if available, in coculture assays. Individual cultures are then rapidly expanded in the presence of excess irradiated
feeder lymphocytes, an antibody targeting the
epsilon subunit within the human CD3 complex of
the TCR, and IL-2. By ~5 to 6 weeks after resecting the tumor, up to 1011 lymphocytes can be obtained for infusion into patients. A substantial
increase in cell persistence and the incidence and
duration of clinical responses was seen when patients received a lymphodepleting preparative
regimen before the cell infusion (13). It might be
possible to optimize the intensity or duration of
the lymphodepletion that is employed, but the
most frequently used lymphodepleting preparative
regimen consists of 60 mg/kg cyclophosphamide
for 2 days and 25 mg/m2 fludarabine adminis-
of 52 and 72% were seen, including 20 and 40%
complete regressions. However, there were no statistically significant differences in the OR rates
between preparative regimens (13, 17). Twenty of
the 93 patients (22%) in these trials had complete
regressions, and 19 (20%) have not experienced
recurrences at follow-up times of 5 to 10 years and
are probably cured. A prospective randomized
study comparing the chemotherapy preparative
regimen alone versus chemotherapy plus the addition of 1200 cGy TBI in 101 patients was recently concluded at the NCI, National Institutes
of Health (NIH), and results are pending.
In the combined experience of the treatment
of 194 patients using TILs grown from individual melanoma fragments at the NCI (Bethesda,
Maryland), 107 patients (55%) have shown ORs.
Similar OR rates to TIL therapy have been reported by multiple groups, including those from
the Moffitt Cancer Center (Tampa, Florida) (38%
OR rate) (18), the MD Anderson Cancer Center
(Houston, Texas) (48% OR rate) (19), and the Ella
Cancer Institute (Ramat Gan, Israel) (40% OR
rate) (20) (Table 1).
There is no relation between the bulk of disease
or the site of metastases and the likelihood of
achieving a complete cancer regression (17). Of the
SCIENCE sciencemag.org
in the NCI trials, OR rates in patients who had no
prior therapy or who progressed through IL-2, antibody to cytotoxic T lymphocyte–associated protein 4 (anti–CTLA-4), anti-PD1, or Braf inhibitors
were 48, 63, 42, 50, and 43%, respectively.
Lymphodepletion appears to be an important
component of ACT, and mouse models have shown
that lymphodepletion given before cell transfer
can increase the effectiveness of treatment more
than 10-fold. In the clinic, the persistence of T cells
was once a rarity (22), but in trials conducted after
the initiation of lymphodepleting therapy, adoptively transferred T cells could comprise the majority of the peripheral blood CD8+ cells 1 month after
transfer (13). The cellular basis of the effect of
lymphodepletion is complex and still not completely understood. In mouse models, myeloid-derived
suppressor cells and CD4+ FoxP3 regulatory T cells
can be found at high levels in tumors in vivo and
can depress immune responses in the mouse tumor microenvironment (23). In accord with these
preclinical findings, preparative chemotherapy in
humans severely depletes lymphocytes and myeloid cells from the circulation at the time of cell
infusion, although the rate of reappearance of
FoxP3 inhibitory T cells after lymphodepletion
was inversely correlated with clinical response (24).
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Table 1. Selected clinical trials of ACT for the treatment of human cancer. CLL, chronic lymphocytic leukemia; ALL, acute lymphocytic leukemia;
CR, complete response; HPC, human papillomavirus; allo-HSCT, allogeneic hematopoietic stem cell transplantation; DLBCL, diffuse large B cell
lymphoma; EBV, Epstein-Barr virus. Dashes indicate not applicable.
CELLS USED FOR ACT
YEAR
CANCER HISTOLOGY
Tumor-inflitrating
lymphocytes*
1998
Melanoma (12)
MOLECULAR TARGET
PATIENTS
NUMBER OF ORS
20
55%
COMMENTS
Original use TIL ACT
............................................................................................................................................................................................................................................................................................................................................
1994
Melanoma (88)
86
34%
2002
Melanoma (13)
13
46%
............................................................................................................................................................................................................................................................................................................................................
Lymphodepletion before cell transfer
............................................................................................................................................................................................................................................................................................................................................
2011
Melanoma (17)
93
56%
20% CR beyond 5 years
............................................................................................................................................................................................................................................................................................................................................
2012
Melanoma (19)
31
48%
............................................................................................................................................................................................................................................................................................................................................
2012
Melanoma (18)
13
38%
Intention to treat: 26% OR rate
............................................................................................................................................................................................................................................................................................................................................
2013
Melanoma (20)
57
40%
Intention to treat: 29% OR rate
2014
Cervical cancer (89)
9
33%
Probably targeting HPV antigens
............................................................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................................................
2014
Bile duct (44)
Mutated ERB2
1
Selected to target a somatic mutation
–
............................................................................................................................................................................................................................................................................................................................................
In vitro sensitization
2008
Melanoma (90)
NY-ESO-1
9
33%
Clones reactive against cancer-testes
antigens
............................................................................................................................................................................................................................................................................................................................................
2014
Leukemia (91)
WT-1
11
–
2010
Lymphoma (16)
CD19
1
100%
Many treated at high risk for relapse
............................................................................................................................................................................................................................................................................................................................................
Genetically engineered
with CARs
First use of anti-CD19 CAR
............................................................................................................................................................................................................................................................................................................................................
2011
CLL (68)
CD19
3
100%
Lentivirus used for transduction
............................................................................................................................................................................................................................................................................................................................................
2013
ALL (70)
CD19
5
100%
Four of five then underwent allo-HSCT
............................................................................................................................................................................................................................................................................................................................................
2014
ALL (92)
CD19
30
90%
CR in 90%
2014
Lymphoma (71)
CD19
15
80%
Four of seven CR in DLBCL
............................................................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................................................
2014
ALL (93)
CD19
16
88%
Many moved to allo-HSCT
............................................................................................................................................................................................................................................................................................................................................
2014
ALL (94)
CD19
21
67%
Dose-escalation study
............................................................................................................................................................................................................................................................................................................................................
27%
CR2 CARs into EBV-reactive cells
2011
Neuroblastoma (78)
GD2
11
............................................................................................................................................................................................................................................................................................................................................
Genetically engineered
with TCRs
2011
Synovial sarcoma (81)
NY-ESO-1
6
67%
First report targeting nonmelanoma
solid tumor
............................................................................................................................................................................................................................................................................................................................................
2006
Melanoma (15, 32)
MART-1
11
45%
............................................................................................................................................................................................................................................................................................................................................
*Molecular targets of TIL in melanoma appear to be exomic mutations expressed by the cancer (39, 40, 44)
Levels of homeostatic cytokines, which promote T
cell proliferation and survival, are dramatically
induced upon lymphodepletion (25) in mouse
models. In humans, lymphodepletion leads to the
appearance in the circulation of the T cell growth
factor IL-15, which serves to promote the expansion
of the transferred cells in the absence of competing
endogenous lymphocytes (26). Further, lymphodepletion can enhance the translocation of commensal microflora across mucosal barriers in the
mouse, and this can enhance the effect of ACT by
stimulating Toll-like receptors (27) to activate
antigen-presenting cells (APCs). These preclinical
results have highly affected clinical translation, and
it seems likely that immune ablation will be a part
of future cell-based treatments in patients with
cancer.
Adoptive cell therapy is a “living” treatment, and
administered lymphocytes can expand more than
1000-fold after administration. Studies in mouse
models, including those involving the injection of
human cells into immunodeficient animals, have
emphasized the importance of the differentiation
state of the infused cells (28, 29). The phenotypic
and functional status of less differentiated murine
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cells is highly positively correlated with their ability to eliminate vascularized tumor in vivo. These
findings are in accordance with the high positive correlation between the persistence of the
transferred TILs in the circulation of patients at
1 month and with the induction of partial and complete clinical responses (17). Further, one clinical
study showed a strong correlation between expression of the phenotypic marker CD27, which is associated with cells early in their differentiation
pathway, and clinical response (17). The presence of
longer telomeres as a correlate of clinical response
was seen in one study (17) but not in another (18).
The observation that melanoma TILs can mediate
durable, complete, and probably curative cancer
regression in patients with metastatic melanoma
has raised considerable interest in the possible use
of TILs for the treatment of multiple cancer types.
Although TILs can be grown in vitro from virtually
all tumors, only melanomas consistently give rise
to TILs with antitumor reactivity. In an attempt to
gain insight into the possible extension of ACT to
the treatment of other common cancers, extensive
studies of the antigens recognized by TILs have
been pursued.
Melanoma TILs recognize the
products of cancer mutations
Early studies identified two nonmutated melanomamelanocyte differentiation proteins, MART-1 and
gp100, that were often recognized by melanoma
TILs (30, 31). Melanocytes in the skin, eye, and
ear express the MART-1 and gp100 proteins, and
yet toxicity targeting these proteins was not
seen in the majority of patients treated with
TILs who underwent complete cancer regression. In contrast, when a high-affinity TCR against
MART-1 or gp100 was inserted into lymphocytes
used for ACT, profound eye and ear toxicity was
often seen in the absence of antitumor activity, which suggests that the reactivity against
melanoma-melanocyte antigens was not the
decisive target resulting in the in vivo antitumor
activity of melanoma TILs (32).
A study of exomic mutation rates in more than
3000 tumor-normal pairs revealed that the frequency of nonsynonymous mutations varied more
than 1000-fold across different cancer types (33).
Pediatric cancers exhibited mutation frequencies as low as 0.1/Mb, whereas melanomas and
lung cancers often exceeded 100 mutations/Mb.
sciencemag.org SCIENCE
The suggestion that mutations might be targets of immune recognition of tumor cells has
been around for some time (34). The responsiveness of melanoma to a variety of immunotherapy approaches such as ACT, IL-2, anti–CTLA-4,
and anti–PD-1 suggested that peptide epitopes
encoded by the large number of mutations in
melanoma might be the targets of TIL therapy
(35). Support for this hypothesis comes from recent observations that anti–PD-1 can mediate
ORs not only in patients with melanoma but
also in patients with lung and bladder cancer,
the two tumor types closest to melanoma with
a high frequency of mutations (36). A patient
successfully treated with anti–CTLA-4 generated circulating T cells that recognized a distinct mutation in the melanoma (37). Another
study suggested that increased numbers of
exomic mutations in a cancer correlated with
better outcomes (38).
New approaches using whole-exomic sequencing of tumor-normal pairs in patients with
melanoma have consistently identified nonsynonymous cancer mutations recognized by
autologous TILs that mediated complete cancer regressions (39, 40). However, not all expressed mutations can be recognized by T cells.
Proteins incorporating the mutations must be
processed to short peptides of ~9 amino acids
for major histocompatibility complex (MHC)
class 1 and a bit longer for MHC class 2; these
peptides are then presented on the cell surface. One approach to identify the immunogenic
mutations that we have taken is to identify
21– to 25–amino acid polypeptides, each one
containing a mutated amino acid flanked by
10 to 12 normal residues. Using peptide-MHC
binding algorithms, these polypeptides can then
be scanned to identify peptides with high binding to individual MHC molecules of the patient.
The top-predicted binding peptides are then
synthesized and tested for recognition by coculture with TILs that mediated cancer regression.
This method depends on the accuracy of peptideMHC binding algorithms, which are often inadequate for many of the less frequent MHC
molecules (39).
An alternate method eliminates the need for
predicted peptide binding to MHC and enables
the screening of all candidate peptides on all
MHC loci in a single test (40) (Fig. 3). As above,
minigenes, rather than polypeptides, are constructed that encode each mutated amino acid
flanked by 10 to 12 amino acids. Strings of 6 to
20 minigenes are then linked into tandem minigenes, and these DNA constructs are subsequently cloned into an expression plasmid and in vitro
transcribed to RNA, which is electroporated into
the patient’s autologous APCs. These APCs present
all mutated peptides capable of being processed
and binding to any of the patient’s class 1 or
class 2 MHC molecules. Culture of the patient’s
TILs with these APCs can identify the tandem
minigene as well as the individual minigene
responsible for tumor recognition. Using these
approaches, TILs from 21 patients with mela-
Tumor sample
Expand and treat
or isolate TCR for insertion
into autologous lymphocytes
Reinfuse post-lymphodepletion
Extract DNA from normal
and malignant tissue
noma that responded to ACT identified 45 mutations presented on a variety of class 1 and
class 2 MHC molecules. Thus far, every mutation recognized by TILs was distinct (i.e., each
from a different expressed protein), with none
shared by another melanoma in the set studied.
These findings provide suggestive evidence that
melanoma TILs capable of mediating antitumor responses were recognizing random somatic mutations in the cancer. In many cases,
multiple mutations were recognized by an individual TIL population. The concept that cancer
regressions after immunotherapy are the result of targeting mutations explains why patients
can experience tumor regression without autoimmune sequelae. Conversely, the ineffectiveness of the vast number of therapeutic cancer
vaccines that targeted nonmutated self-proteins
can also be explained (41, 42). Whereas strong
reactivity to self-antigens causes autoimmune
toxicity, vaccines against self-antigens trigger
the expansion of low-affinity TCRs against selfproteins that escaped negative selection in the
thymus. This raises the possibility that vaccines
targeting mutated immunogenic epitopes may
be much more effective. The specific targeting
of individual mutated antigens in a patient’s cancer presents a daunting problem for widespread
therapeutic application of ACT but also presents
an opportunity to develop treatments for multiple
cancer types. Schumacher and Schreiber discuss
additional aspects for targeting mutated antigens
in this issue (43).
T cells from peripheral
blood or tumor
T cells
Exome sequencing to
identify cancer mutations
Some mutations
are presented by APC
in the context of
autologous MHC
Co-culture T cells
41BB or
OX40
MHC
TCR
APC
α
Select using
activation marker
β
DSSLQARLFPGLTIKIQRSNGLIHS
Tandem minigenes or synthetic peptides
of all mutations introduced to autologous APC
Fig. 3. A “blueprint” for the treatment of patients with T cells recognizing tumor-specific mutations. The sequences of exomic DNA from tumor
cells and normal cells from the same patient are compared to identify tumorspecific mutations. Knowledge of these mutations can then be used to
synthesize either minigenes or polypeptides encoding each mutated amino
acid flanked by 10 to 12 amino acids. These peptides or minigenes can be
expressed by a patient’s autologous APCs, where they are processed
and presented in the context of a patient’s MHC. Coculture of the patient’s
SCIENCE sciencemag.org
Activation
marker
T cells with these APCs can be used to identify all mutations processed
and presented in the context of all of a patient’s MHC class I and class II
molecules. The identification of individual mutations responsible for tumor
recognition is possible because T cells express activation markers, such as
41BB (CD8+ T cells) and OX40 (CD4+ T cells), when they recognize their
cognate target antigen. T cells expressing the activation marker can then
by purified using flow cytometry before their expansion and reinfusion into
the tumor-bearing patient.
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TILs from common epithelial cancers
can also recognize cancer mutations
A recent report has shown that the mutated
antigens in a nonmelanoma epithelial cancer
can give rise to immune responses, despite the
low number of mutations in these cancers (44).
Exomic sequencing of a metastatic cholangiocarcinoma in a patient who had progressed
through multiple chemotherapies revealed 26
nonsynonymous mutations. Tandem minigenes
that encoded each mutated amino acid and its
flanking sequences were constructed and electroporated into the patient’s APCs. CD4 cells from
TIL cultures from this patient’s tumor recognized
the ERBB2IP mutation restricted by the MHC
class 2 antigen HLA-DQ O6. ERBB2IP is a tumor
suppressor that binds to ERBB2 and attenuates
downstream RAS/ERK signaling. Despite the lack
of an objective clinical response to the administration of bulk autologous TILs in this patient,
administration of TILs that were selected to contain more than 95% ERBB2IP mutation-reactive
TILs mediated a dramatic regression of liver and
lung metastases ongoing beyond 1 year. This result provides compelling evidence that mutationreactive T cells are capable of mediating in vivo
tumor regression in patients with this epithelial
cancer. Further, the findings suggest that this
treatment approach may be suitable for patients
with other common epithelial cancers that are
not normally considered to be immunogenic.
Mutations that are targeted may be driver
mutations essential for the malignant phenotype
of the cell, or alternatively, the TILs may contain
reactivity against multiple immunogenic passenger
mutations, which would decrease the likelihood
that the loss of any individual antigen would subvert the clinical antitumor response. TIL populations can be highly polyclonal and thus are likely
to be capable of potentially recognizing multiple
antigens simultaneously. Given their curative potential, it seems likely that TILs are able to recognize antigens expressed by cancer stem cells.
Although some of the mutations are probably
driver mutations because they are found in expressed genes associated with known oncogenic
pathways (e.g., mutated b-catenin), many of the
targets of TILs may well be passenger mutations.
Genetic engineering of lymphocytes
for use in ACT
In an attempt to broaden the reach of ACT to
other cancers, techniques were developed to introduce antitumor receptors into normal T cells
that could be used for therapy (Fig. 4). The specificity of T cells can be redirected by the integration of genes encoding either conventional
alpha-beta TCRs or CARs. CARs were pioneered
by Gross and colleagues in the late 1980s (45) and
can be constructed by linking the variable regions
of the antibody heavy and light chains to intracellular signaling chains such as CD3-zeta, often in-
T cell
cluding costimulatory domains encoding CD28
(46) or CD137 to fully activate T cells (47, 48). CARs
can provide non-MHC–restricted recognition of
cell surface components and can be introduced
into T cells with high efficiency using viral vectors.
An important question confronting the use of
genetically engineered cells in the ACT of cancer
involves selection of the ideal human T cell subpopulation into which the gene should be introduced, as well as the selection of appropriate
antigenic targets of the introduced TCRs or CARs.
Preclinical studies in mouse models strongly suggest that improved antitumor responses are seen
when T cells in early stages of differentiation (such
as naïve or central memory cells) are transduced
(49), a result supported by studies in monkeys
showing improved in vivo persistence of infused
central memory compared with effector memory
cells (50). CD8+ T cells can be categorized into distinct memory subsets based on their differentiation states. We and others have found that CD8+
T cells follow a progressive pathway of differentiation from naïve T cells into central memory and
effector memory T cell populations [summarized
in (51)]. CD8+ T cells paradoxically lose antitumor
T cell functionality as they acquire the ability
to lyse target cells and to produce the cytokine
interferon-g, qualities thought to be important in
their antitumor efficacy (52). The differentiation
state of CD8+ T cells is inversely related to their
capacity to proliferate and persist (52–54). These
Preconditioning
with chemotherapy
T cell
receptor
(TCR)
Tumor cell
MHC
T cells from
peripheral
blood
Antigen
processed and
presented by MHC
Viral or
non-viral
insertion of
genes into
T cells
Chimeric
antigen receptor
Tumor cell
(CAR)
Expand
TCR geneengineered
T cells
Cell
infusion
with IL-2
T cell
Antigen
expressed on
the cell surface
Fig. 4. Gene-modification of peripheral blood lymphocytes. In an attempt
to broaden the reach of ACT to other cancers, techniques are being developed
to introduce antitumor receptors into normal T cells that could be used for
therapy. The top panel shows the insertion of a conventional TCR into a
patient’s T lymphocytes, followed by the expansion and infusion back into the
patient. The bottom panel shows the insertion of a CAR into a patient’s T cell,
followed by the expansion of these cells and their re-infusion. TCRs and CARs
are fundamentally different in their structures and in the structures that they
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Preconditioning
with chemotherapy
recognize. TCRs are composed of one a chain and one b chain, and they
recognize antigens that have been processed and presented by one of the
patient’s own MHC molecules. CARs are artificial receptors that can be
constructed by linking the variable regions of the antibody heavy and light
chains to intracellular signaling chains (such as CD3-zeta, CD28, 41BB) alone
or in combination with other signaling moieties. CARs recognize antigens that
do not need to be MHC-restricted, but they must be presented on the tumor
cell surface.
sciencemag.org SCIENCE
findings may be clinically relevant, and younger
T cells are statistically positively correlated with
clinical effectiveness in ACT trials (17). It seems
clear that, like many organ systems in the body,
CD8+ T cells can exist in a stem cell–like state,
capable of clonal repopulation. Human T memory
stem cells express a gene program that enables
them to proliferate extensively and can further
differentiate into other T cell populations (29).
Much of the existing work in cancer immunotherapy has focused on CD8+ T cells. However,
CD4+ T cells can also efficiently promote tumor
rejection. CD4+ T cells do not merely enhance
CD8+ T cell function, but they also play a more
direct role in tumor elimination. This notion has
been validated recently in humans (44). The roles
that CD4+ T cells play in the antitumor immune
response crucially depend on their polarization,
which is determined by their expression of key
transcription factors. CD4+ cells can destroy tumor cells, and recent evidence suggests that adoptively transferred T helper 17 cells can promote
long-lived antitumor immunity (55).
Toxicity of ACT when targeting antigens
shared by tumors and normal tissue
The marked potency of T cells enables the recognition of minute levels of antigen expressed on
normal cells. Thus, targeting normal, nonmutated
antigenic targets that are expressed on normal
tissues but overexpressed on tumors has led to
severe on-target, off-tumor toxicity in patients.
Suitable antigens to target are those presented
exclusively on the cancer or, alternatively, on normal cells that are not essential for survival.
The first successful application of ACT using
genetically engineered lymphocytes treated 17 patients with metastatic melanoma using autologous
T cells transduced with a weakly avid human TCR
recognizing the MART-1 melanoma-melanocyte
differentiation antigen (15). Two patients experienced objective partial regressions of metastatic
melanoma, and in both patients the transferred
cells could be found in the peripheral blood 1 year
after cell infusion. This approach was expanded
to 36 patients with metastatic melanoma who
received high-avidity TCRs that recognized either
the MART-1 or gp100 melanoma-melanocyte antigens (32). Although objective cancer regressions
were observed in 30 and 19% of patients who
received the MART-1 or gp100 TCR, respectively,
severe off-tumor, on-target toxicity was seen in
the skin, eyes, and ears of patients due to the expression of melanocytes in these organs. These
findings coincided with severe eye toxicity seen
in mice when targeting melanocyte antigens and
provided an early demonstration of the power
of T cell therapy (56). The treatment of patients
with renal cancer using T cells encoding a CAR
against carbonic anhydrase 9, which is overexpressed in renal cancer, led to severe liver toxicity due to expression of this antigen in biliary
duct epithelium (57). A high-affinity TCR against
the carcinoembryonic antigen was used to treat
patients with metastatic colorectal cancer that
expressed high levels of this antigen (58). All
three patients experienced life-threatening coliSCIENCE sciencemag.org
tis and colonic hemorrhage that precluded further use of this TCR, even though one patient
exhibited a partial response of liver metastases.
Unexpected toxicities can also result when previously unknown cross-reactivities are seen that
target normal self-proteins expressed in vital
organs. MAGE-A3, a cancer-testes antigen to be
discussed in more detail below, is not known to
be expressed in any normal tissues. However,
targeting an HLA-A*0201–restricted peptide in
MAGE-A3 caused severe damage to gray matter
in the brain, resulting in two deaths because
this TCR recognized a different but related epitope expressed by MAGE-A12, expressed at very
low levels in the brain (59). It should also be
noted that CARs are capable of toxicity against
self-antigens as well. Acute pulmonary toxicity
resulting in death was observed after infusion of
CAR T cells specific for ERBB2, which seemed
likely due to the recognition of low levels of this
antigen on pulmonary epithelium (60).
Several groups have attempted to affinityenhance TCRs by altering amino acids in the
antigen-combining sites of the TCR (61, 62). By
removing the protective effects of negative thymic selection that eliminate high-affinity TCRs
against normal proteins, these modified TCRs
could potentially recognize new and unrelated
determinants. Two patients (one with multiple
myeloma and one with melanoma) were treated
with an HLA-A1–restricted MAGE-A3–specific
TCR whose affinity was enhanced by this sitespecific mutagenesis, and both experienced fatal
cardiogenic shock due to the recognition of an
HLA-A1–restricted peptide derived from an unrelated protein, titin, present in cardiac muscle
(63). Thus, methods aimed at enhancing the affinities of TCRs can be fraught with problems of
unexpected toxicities, which remain difficult to
predict. Of course, the same pitfalls of unexpected
toxicities may apply to the use of novel CARs.
Targeting antigens expressed on cancers
and nonessential human tissues
Cancers that express target molecules shared with
nonessential normal organs represent potential
targets for human cancer immunotherapy using
ACT. A prominent example of such an antigen is
the CD19 molecule expressed on more than 90% of
B cell malignancies and on B cells at all stages of
differentiation, excluding plasma cells. Following
preclinical work by many groups [summarized in
(64–67)], the first successful clinical application
of anti-CD19 CAR gene therapy in humans was
reported in 2010 (16). Administration of autologous
cells expressing the anti-CD19 CAR to a patient with
refractory lymphoma resulted in cancer regression
in a patient who remains progression-free after
two cycles of treatment ongoing 4 years after treatment. Multiple groups have now shown the effectiveness of ACT targeting CD19 in patients with
follicular lymphoma, large-cell lymphomas, chronic lymphocytic leukemia, and acute lymphocytic
leukemia (68–72). On-target toxicity against CD19
results in B cell loss in the circulation and in the
bone marrow and can be overcome by the periodic
administration of immunoglobulin infusions. Sub-
stantial toxicity can be seen by the excessive release
of cytokines by CAR-expressing cells, and thus, careful selection of the lymphodepleting preparative
regimen and the cell dose is required to safely apply
ACT targeting CD19, as well as many other antigens now under experimental study (72).
Dramatic regressions of lymphomas and leukemias with ACT have elicited considerable enthusiasm, although most reports contain fewer than
20 patients, and fewer than 200 patients have been
treated worldwide. The introduction of CARs into
lymphocytes has mainly used gammaretroviruses
and lentiviruses, although nonviral approaches such
as transposon-transposase systems (73) and CRISPRcas (CRISPR, clustered regularly interspaced palindromic repeat) technology to introduce genes are
also being explored (74). The single-chain antibody governs recognition of the antigen to be
targeted, although the T cell is activated via the
CD3-zeta chain signaling domain. In addition to
the zeta chain, a variety of costimulatory molecules
have been employed in retroviral constructs such
as CD27, CD28, CD134, CD137, or ICOS that can
profoundly influence the function of the CAR
[reviewed in (64–66)]. Optimization of these costimulatory domains is a subject of active study.
The results of CAR therapy for B cell malignancies might be confounded by the sensitivity
of lymphomas and leukemias to the preparative
chemotherapy regimen. Thus, delineation between
the effects of the preparative therapy and those
of the CAR T cells needs to be considered.
Multiple other B cell antigens are being studied
as targets, including CD22, CD23, ROR-1, and the
immunoglobulin light-chain idiotype expressed by
the individual cancer (65). CARs targeting either
CD33 or CD123 have been studied as a therapy for
patients with acute myeloid leukemia, though the
expression of these molecules on normal precursors can lead to prolonged myeloablation (75).
BCMA is a tumor necrosis factor receptor family
protein expressed on mature B cells and plasma
cells and can be targeted on multiple myeloma (65).
The Reed-Sternberg cell expresses CD30, and this
target is being explored as a treatment for patients with refractory Hodgkin lymphoma (75–77).
Although CARs are being successfully applied
to the treatment of hematologic malignancies, the
lack of shared antigens on the surface of solid tumors that are not also expressed on essential normal tissues has severely limited the application of
CARs to the treatment of solid tumors. Thyroglobulin is a potential target for some patients with
thyroid cancers because thyroglobulin is present
only in the thyroid gland and not on solid tissues.
Neuroblastomas express GD2, which has been
targeted by CARs (78). Mesothelin has also been
forwarded as a potential target, although it is
also expressed on normal tissues, including cells
in the pericardium and pleural and pertitoneal
linings (79). A search is ongoing for other tissuespecific surface antigens expressed on tissues that
are not essential for survival.
Cancer-testis antigens are a family of intracellular proteins that are expressed during fetal development but have highly restricted expression
in adult normal tissues (80). There are more than
3 APRIL 2015 • VOL 348 ISSUE 6230
67
C A NC E R IM M UN OL OG Y A N D I MM U NO TH E R AP Y
100 different members of this family of molecules
whose expression is epigenetically up-regulated
from 10 to 80% of cancer types using highly sensitive techniques. However, initial enthusiasm for
targeting cancer-testes antigens has been tempered
by the lack of high levels of protein expression
of these antigens. Approximately 10% of common
cancers appear to express enough protein to be
suitable targets for antitumor T cells. There are
low levels of some cancer-testes antigens expressed
on normal tissues, and this can lead to untoward
toxicities. The NYESO-1 cancer-testes antigen has
been targeted via a human TCR transduced into
autologous cells (81). ORs were seen in 5 of 11 patients with metastatic melanoma and 4 of 6 patients with highly refractory synovial cell sarcoma.
Looking to the future of ACT for
the treatment of cancer
The continued development of ACT, as well as
other immunologic approaches to the treatment
of cancer, depends on the identification of suitable targets for immunologic attack. Although
CARs have been successful in the treatment of
hematologic malignancies and are likely to soon
join the mainstream of oncologic treatment, the
ability to treat common epithelial solid cancers,
which account for ~90% of all cancer fatalities,
is severely limited by the lack of suitable targets
exclusive to cancer. Extensive searches for monoclonal antibodies that can recognize distinct determinants on the surface of solid cancers but
not normal tissues have been in progress for more
than 30 years, but few suitable determinants have
been found. The EGFRvIII mutation on ~40% of
high-grade glioblastomas is a rare example of a
shared-surface mutation, and attempts to target
this molecule using CARs are in progress (82).
Shared mutations in intracellular proteins involved in oncogenesis—such as Braf in melanomas and Kras in pancreatic and other solid
cancers—would be ideal ACT targets using conventional alpha-beta TCRs, though immunogenic
epitopes have not yet been identified in these
molecules. Driver and random somatic mutations
occurring in many solid cancers may represent
excellent targets for the treatment of solid tumors.
Opportunities to improve ACT involve the identification and development of specific antitumor
T cells with the functional properties optimal
for tumor destruction (83). One approach under
active evaluation is the growth of cells under conditions that enable in vitro proliferation while
limiting differentiation, such as the use of IL-21
or inhibitors that target the kinase AKT (84, 85).
Improved specific lymphodepleting preparative
regimens and better design of the transducing
vectors, including the incorporation of optimal
costimulatory molecules, are likely to improve
clinical results. Introduction of genes encoding
other molecules such as the cytokine IL-12, which
can profoundly alter the tumor microenvironment to favor antitumor immunity, has shown
substantial promise in animal models (86). Enhanced methods for regulating the expression of
these highly potent cytokine genes would be an
important part of incorporating them into clinical
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3 APRIL 2015 • VOL 348 ISSUE 6230
treatment. The incorporation of “suicide” genes
that can enable destruction of the transferred cells
could add an extra level of safety when exploring genetic changes in lymphocytes (87).
Adoptive cell therapy is a more complex approach to the delivery of cancer treatment than
many other types of immunotherapy and has
often been criticized as impractical and too costly
for widespread application. The need to develop
highly personalized treatments for each patient
does not fit into the paradigm of major pharmaceutical companies that depend on “off-the-shelf ”
reagents that can be widely distributed. However, curative immunotherapies for patients with
common epithelial cancers will probably dictate
the need for more personalized approaches. Several new biotechnology companies have arisen to
meet the need to expand a patient’s lymphocytes,
and detailed genetic analysis of individual tumors
is already commonplace at large academically affiliated medical centers. Although multiple commercial models have been proposed, widespread
application of ACT will probably depend on the
development of centralized facilities for producing tumor-reactive TILs or genetically modified
lymphocytes that can then be delivered to the
treating institution. The effectiveness of treatment
will need to trump convenience of administration
in the application of new effective approaches to
cancer immunotherapy.
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AC KNOWLED GME NTS
We thank the Center for Cancer Research, NCI, NIH, Bethesda,
Maryland, and the Milstein Family Foundation for their generous
support.
10.1126/science.aaa4967
sciencemag.org SCIENCE
Adoptive cell transfer as personalized immunotherapy for human
cancer
Steven A. Rosenberg and Nicholas P. Restifo
Science 348, 62 (2015);
DOI: 10.1126/science.aaa4967
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